Acharya and Lau-Cam Journal of Biomedical Science 2010, 17(Suppl 1):S35 http://www.jbiomedsci.com/content/17/S1/S35 REVIEW Open Access Comparison of the protective actions of N-acetylcysteine, hypotaurine and taurine against acetaminophen-induced hepatotoxicity in the rat Miteshkumar Acharya†, Cesar A Lau-Cam* From 17th International Meeting of Taurine Fort Lauderdale, FL, USA. 14-19 December 2009 Abstract When used in overdoses, acetaminophen (APAP) is a common cause of morbidity and mortality in humans. At pre- sent, N-acetylcysteine (NAC) is the antidote of choice for acetaminophen overdoses. Prompt administration of NAC can prevent the deleterious actions of APAP in the liver. In view of the similarities in antioxidant effects demon- strated by NAC, hypotaurine (HYTAU) and taurine (TAU) in this and other our laboratories, the present study was undertaken to compare these compounds for the ability to attenuate plasma and liver biochemical changes asso- ciated with a toxic dose of APAP. For this purpose, fasted male Sprague-Dawley rats, 225-250 g in weight, were intraperitoneally treated with APAP (800 mg/kg), NAC, HYTAU or TAU (2.4 mM/kg) followed 30 min later by APAP, or 50% PEG 400 (the vehicle for APAP). At 6 hr after APAP administration, all animals were sacrificed by decapita- tion and their blood and livers collected. The plasma fractions were analyzed for indices of liver damage (alanine transaminase, aspartate transaminase, lactate dehydrogenase), levels of malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione, and activities of glutathione reductase (GR), glutathione S-transferase (GST) and g-glu- tamylcisteinyl synthetase (GCS). Suitable liver homogenates were analyzed for the same biochemical parameters as the plasma but indices of liver damage. By itself, APAP increased MDA formation and had a significant lowering influence on the levels of GSH and GSSG, the GSH/GSSH ratio, and the activities of GR, GST and GCS both in the plasma and liver. In addition, APAP promoted the leakage of transaminases and lactate dehydrogenase from the liver into the plasma. Without exceptions, a pretreatment with a sulfur-containing compound led to a significant attenuation of the liver injury and the biochemical changes induced by APAP. Within a narrow range of potency differences, HYTAU appeared to be the most protective and TAU the least. The present results suggest that, irre- spective of the differences in structural features and in vitro antioxidant properties that may exist among NAC, TAU and HYTAU, these compounds demonstrate equivalent patterns of protection and, to a certain extent, equipotent protective actions against the toxic actions of APAP in the liver when tested in equimolar doses and under the same conditions in an animal model. * Correspondence: [email protected] † Contributed equally Department of Pharmaceutical Sciences, St. John’s University, College of Pharmacy and Allied Health Professions, 8000 Utopia Parkway, Jamaica, New York 11439, USA Full list of author information is available at the end of the article © 2010 Lau-Cam and Acharya; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acharya and Lau-Cam Journal of Biomedical Science 2010, 17(Suppl 1):S35 Page 2 of 11 http://www.jbiomedsci.com/content/17/S1/S35 Background immediate metabolic precursor of TAU, with that of Therapeutic doses of the analgesic drug acetaminophen N-acetyl-L-cysteine (NAC), an L-cysteine analog that (APAP) are readily detoxified by hepatic phase II drug- can serve as a substrate for GSH synthesis [16,18,25]. metabolizing systems mediating glucuronidation and sul- While NAC is regarded as the antidote of choice for fation [1], with a small portion undergoing a cytochrome APAP overdoses [18] and as an effective antioxidant P-450-mediated bioactivation to the highly reactive elec- [26-28], information on the protective actions of TAU trophilic arylating intermediate N-acetyl-p-benzoquino- in APA-induced acute liver injury appears to be limited neimine (NAPQI) [2]. In rats and humans, NAPQI is to a report indicating that this amino acid can attenuate detoxified principally by conjugation with reduced the leakage of intracellular enzymes from and DNA glutathione (GSH) under spontaneous or glutathione S- fragmentation, lipid peroxidation (LPO), apoptosis and transferase (GST)-mediated conditions to the 3-glu- necrosis in the hepatocytes of rats receiving a toxic dose tathione-S-yl-APAP conjugate [1]. of APAP [29]. On the other hand, earlier work from this In the event of the intake of an overdose of APAP, the laboratory has determined that both TAU) and HYTAU increased production of NAPQI rapidly overwhelms can attenuate LPO, preserve the intracellular stores of GST, eventually exhausts GSH, UDP-glucuronic acid GSH, and prevent the losses of antioxidant enzyme and inorganic sulfate [3], inhibits GSH synthesis [3,4] activities in erythrocytes of rats exposed to an oxidant and decreases cytosolic GST activity [5]. More impor- [30] or affected by type 2 diabetes [31]. In addition, the tantly, this APAP metabolite is a major cause of hepato- results of in vitro experiments with free radical generat- cellular damage, centrilobular hepatic necrosis and even ing systems have suggested that HYTAU, because of its fatalities upon entering in adduct formation with liver sulfinate functionality, is a better antioxidant and radical macromolecules, especially proteins [6]. scavenger than TAU [32]. The purpose of the present The hepatotoxicity of APAP is generally recognized to study was to compare NAC, HYTAU and TAU for their start with the formation of NAPQI and to be related to effects in preventing oxidative stress and, thus, liver the oxidative stress that develops as a result of the oxi- injury, when administered in equimolar doses to rats dative capacities of this reactive metabolic product and as a pretreatment to a toxic dose of APAP. [6-10]. NAPQI is capable of lowering GSH/GSSG ratio by oxidizing the thiol group of GSH and of promoting Methods the formation of interstrand disulfide linkages, interpro- Animals tein cross links and protein-GSH mixed disulfides by Male Sprague-Dawley rats, 225-250 g in weight, were pur- oxidizing cysteine thiol groups in proteins [10]. APAP chased from Taconic Farms, Germantown, New York, may also cause hepatotoxicity by mechanisms leading to USA, and housed in a temperature controlled room (21 the formation of reactive oxygen species (ROS), such as ±1°C) with a 12 hr light-12 hr dark cycle; and allowed free -• superoxide anion (O2 ), hydrogen peroxide (H2O2)and access to a standard rat chow and filtered tap water for at hydroxyl radical (HO•), reactive nitrogen species (RNS), least 5 days. The solid food, but not the water, was such as nitric oxide and peroxynitrite (ONOO-), and removed 12 hr prior to an experiment. The study received peroxidation reaction products [6-8,10]. the approval of the Institutional Animal Care and Use Furthermore, the APAP-associated depletion of the Committee of St. John’s University, and the animals were intrahepatic GSH is accompanied by variable alterations cared for in accordance with the guidelines established by in glutathione disulfide (GSSG) contents [11,12] and by the United States Department of Agriculture. reductions in the activities of the antioxidant enzymes glutathione reductase (GR) [13,14], g-glutamylcysteinyl Treatments and samples synthetase (GCS), catalase (CAT), glutathione peroxi- The treatment solutions were prepared either in warm dase (GPX) and superoxide dismutase (SOD) [14]. 50% PEG 400 (APAP) or in distilled water (NAC, TAU, Since NAPQI formation, GSH depletion, and the alky- HYTAU). All the solutions were administered by the lation of proteins, especially in the mitochondrion, are intraperitoneal (i.p.) route. A treatment compound was central to the onset of hepatotoxicity by APAP [15], administered as a single, 2.4 mM/kg/2 mL dose, 30 min compounds with potential for serving as a source of before APAP (800 mg/kg/2 mL). Control animals GSH [3,16-19] or for preventing oxidative reactions received only 50% PEG 400 or only a sulfur-containing [19-24] have been extensively studied for the ability to compound. The animals were sacrificed by decapitation counteract APAP toxicity. Based on this evidence, the at 6 hr post-APAP, and their blood samples were present study was aimed at comparing the hepatopro- promptly delivered into heparinized test tubes for subse- tective actions of taurine (TAU), a nonprotein sulfur- quent centrifugation and processing for the plasma frac- containing amino acid, and hypotaurine (HYTAU), the tion. Following surgery of the animals, the livers were Acharya and Lau-Cam Journal of Biomedical Science 2010, 17(Suppl 1):S35 Page 3 of 11 http://www.jbiomedsci.com/content/17/S1/S35 exposed and immediately freeze-clamped with metal hydroxide in a ratio of 1:6, and an aliquot of this dilution tongs precooled in liquid nitrogen, and stored in liquid was treated with an equal volume of OPT. The fluores- nitrogen until needed. cence of the dilution was read on a fluorometer as described for GSH. The concentrations of GSH and Liver homogenates GSSG in the sample were derived by reference to calibra- A liver homogenate for the assay of GSH and GSSG was tion curves of GSH and GSSG prepared from serial dilu- prepared by mixing a portion of liver sample with 4 tions of GSH and GSSG stock solutions that were treated times its volume of 25% metaphosphoric acid plus 14 in an identical manner as the lung homogenate or plasma times its volume of PBS pH 8.0, followed by homogeni- sample. The results were reported as µM/g of tissue in zation on ice with the help of a hand held electric blen- the case of liver samples and as µg/ml in the case of der.